EP2501817B1 - Maus mit gemeinen leichten ketten - Google Patents

Maus mit gemeinen leichten ketten Download PDF

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Publication number
EP2501817B1
EP2501817B1 EP11703799.4A EP11703799A EP2501817B1 EP 2501817 B1 EP2501817 B1 EP 2501817B1 EP 11703799 A EP11703799 A EP 11703799A EP 2501817 B1 EP2501817 B1 EP 2501817B1
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EP
European Patent Office
Prior art keywords
human
mouse
heavy chain
gene
light chain
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP11703799.4A
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English (en)
French (fr)
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EP2501817A1 (de
EP2501817B2 (de
Inventor
John Mcwhirter
Lynn Macdonald
Sean Stevens
Samuel Davis
Andrew J. Murphy
David R. Buckler
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=43663643&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP2501817(B1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to PL11703799T priority Critical patent/PL2501817T5/pl
Priority to PL12173456T priority patent/PL2505654T5/pl
Priority to SI201130642T priority patent/SI2501817T2/sl
Priority to DK12173456.0T priority patent/DK2505654T4/da
Priority to EP12173456.0A priority patent/EP2505654B2/de
Priority to MEP-2015-197A priority patent/ME02288B/me
Priority to RS20150772A priority patent/RS54367B2/sr
Priority to EP16178223.0A priority patent/EP3095871B1/de
Priority to EP19157278.3A priority patent/EP3540066A1/de
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to PL16178223T priority patent/PL3095871T3/pl
Publication of EP2501817A1 publication Critical patent/EP2501817A1/de
Publication of EP2501817B1 publication Critical patent/EP2501817B1/de
Priority to HRP20151188TT priority patent/HRP20151188T4/hr
Priority to CY20151101020T priority patent/CY1116922T1/el
Priority to CY20161101191T priority patent/CY1118241T1/el
Priority to SM201600432T priority patent/SMT201600432B/it
Priority to CY20191100710T priority patent/CY1121794T1/el
Publication of EP2501817B2 publication Critical patent/EP2501817B2/de
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    • A—HUMAN NECESSITIES
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    • A01K2217/00—Genetically modified animals
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/567—Framework region [FR]
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Definitions

  • a genetically modified mouse that expresses antibodies having a common human variable/mouse constant light chain associated with diverse human variable/mouse constant heavy chains.
  • a method for making a human bispecific antibody from human variable region gene sequences of B cells of the mouse is provided.
  • Antibodies typically comprise a homodimeric heavy chain component, wherein each heavy chain monomer is associated with an identical light chain.
  • Antibodies having a heterodimeric heavy chain component e.g., bispecific antibodies
  • bispecific antibodies are desirable as therapeutic antibodies. But making bispecific antibodies having a suitable light chain component that can satisfactorily associate with each of the heavy chains of a bispecific antibody has proved problematic.
  • a light chain might be selected by surveying usage statistics for all light chain variable domains, identifying the most frequently employed light chain in human antibodies, and pairing that light chain in vitro with the two heavy chains of differing specificity.
  • a light chain might be selected by observing light chain sequences in a phage display library (e.g ., a phage display library comprising human light chain variable region sequences, e.g ., a human ScFv library) and selecting the most commonly used light chain variable region from the library. The light chain can then be tested on the two different heavy chains of interest.
  • a phage display library e.g ., a phage display library comprising human light chain variable region sequences, e.g ., a human ScFv library
  • a light chain might be selected by assaying a phage display library of light chain variable sequences using the heavy chain variable sequences of both heavy chains of interest as probes.
  • a light chain that associates with both heavy chain variable sequences might be selected as a light chain for the heavy chains.
  • a candidate light chain might be aligned with the heavy chains' cognate light chains, and modifications are made in the light chain to more closely match sequence characteristics common to the cognate light chains of both heavy chains. If the chances of immunogenicity need to be minimized, the modifications preferably result in sequences that are present in known human light chain sequences, such that proteolytic processing is unlikely to generate a T cell epitope based on parameters and methods known in the art for assessing the likelihood of immunogenicity (i.e., in silico as well as wet assays).
  • mice that express human immunoglobulin heavy and light chain variable domains, wherein the mice have a limited light chain variable repertoire.
  • a mouse is provided where the mouse is one comprising
  • the genetically engineered mice provided may select suitable affinity-matured human immunoglobulin heavy chain variable domains derived from a repertoire of unrearranged human heavy chain variable region gene segments, wherein the affinity-matured human heavy chain variable domains associate and express with a single human light chain variable domain derived from one human light chain variable region gene segment.
  • mice express a single human light chain variable domain, from a single human light chain variable region gene segments.
  • a genetically modified mouse that comprises a single human immunoglobulin light chain variable (VL) region gene segment that is capable of rearranging and encoding a human VL domain of an immunoglobulin light chain.
  • VL light chain variable
  • the mouse comprises no more than two human VL gene segments that are capable of rearranging and encoding a human VL domain of an immunoglobulin light chain.
  • the genetically modified mouse comprises a single rearranged (V/J) human immunoglobulin light chain variable (VL) region segment (i.e., a V/J segment) that encodes a human VL domain of an immunoglobulin light chain.
  • V/J human immunoglobulin light chain variable
  • the VL gene segment is a human V ⁇ 1-39J ⁇ 5 gene segment or a human V ⁇ 3-20J ⁇ 1 gene segment.
  • the human VL gene segment is operably linked to a human or mouse leader sequence.
  • the leader sequence is a mouse leader sequence.
  • the mouse leader sequence is a mouse V ⁇ 3-7 leader sequence.
  • the VL gene segment is operably linked to an immunoglobulin promoter sequence.
  • the promoter sequence is a human promoter sequence.
  • the human immunoglobulin promoter is a V ⁇ 3-15 promoter.
  • a genetically modified mouse that comprises a VL locus that does not comprise an endogenous mouse VL gene segment that is capable of rearranging to form an immunoglobulin light chain gene, wherein the VL locus comprises a single human VL gene segment that is capable of rearranging to encode a VL region of a light chain gene.
  • the human VL gene segment is a human V ⁇ 1-39J ⁇ 5 gene segment or a human V ⁇ 3-20J ⁇ 1 gene segment.
  • the VL locus comprises a leader sequence flanked 5' (with respect to transcriptional direction of the VL gene segment) with a human immunoglobulin promoter and flanked 3' with a human VL gene segment that rearranges and encodes VL domain of a reverse chimeric light chain comprising an endogenous mouse light chain constant region (CL).
  • the VL gene segment is at the mouse kappa ( ⁇ ) VL locus, and the mouse CL is a mouse ⁇ CL.
  • the mouse comprises a nonfunctional lambda ( ⁇ ) immunoglobulin light chain locus.
  • the ⁇ locus comprises a deletion of one or more sequences of the locus, wherein the one or more deletions renders the ⁇ locus incapable of rearranging to form a light chain gene.
  • all or substantially all of the VL gene segments of the ⁇ locus are deleted.
  • the VL locus of the modified mouse is a ⁇ locus
  • the ⁇ locus comprises a mouse ⁇ intronic enhancer, a mouse ⁇ 3' enhancer, or both an intronic enhancer and a 3' enhancer.
  • mouse makes a light chain that comprises a somatically mutated VL domain derived from the human VL gene segment.
  • the light chain comprises a somatically mutated VL domain derived from the human VL gene segment, and a mouse ⁇ CL region.
  • the mouse does not express a ⁇ light chain.
  • the genetically modified mouse is capable of somatically hypermutating the human VL region sequence.
  • the mouse comprises a cell that expresses a light chain comprising a somatically mutated human VL domain linked to a mouse ⁇ CL, wherein the light chain associates with a heavy chain comprising a somatically mutated VH domain derived from a human VH gene segment and wherein the heavy chain comprises a mouse heavy chain constant region (CH).
  • a light chain comprising a somatically mutated human VL domain linked to a mouse ⁇ CL
  • the heavy chain associates with a heavy chain comprising a somatically mutated VH domain derived from a human VH gene segment and wherein the heavy chain comprises a mouse heavy chain constant region (CH).
  • CH mouse heavy chain constant region
  • the mouse comprises a replacement of 90-100% of the endogenous mouse heavy chain variable region gene locus with a plurality of human heavy chain variable region gene segments, wherein the human heavy chain variable region gene segments are operably linked to an endogenous mouse heavy chain constant gene, and the human heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric heavy chain gene.
  • the mouse rearranges the human VH gene segments and expresses a reverse chimeric immunoglobulin heavy chain that comprises a human VH domain and a mouse CH.
  • all or substantially all of the endogenous mouse VH gene segments are replaced with a plurality of unrearranged human VH gene segment.
  • the replacement is with at least 19, at least 39, or at least 80 or 81 unrearranged human VH gene segments. In one embodiment, the replacement is with at least 12 functional unrearranged human VH gene segments, at least 25 functional unrearranged human VH gene segments, or at least 43 functional unrearranged human VH gene segments.
  • the mouse comprises a replacement of all mouse D and J segments with at least one unrearranged human D segment and at least one unrearranged human J segment. In one embodiment, the at least one unrearranged human D segment is selected from D1-7, D1-26, D3-3, D3-10, D3-16, D3-22, D5-5, D5-12, D6-6, D6-13, D7-27, and a combination thereof.
  • the at least one unrearranged human J segment is selected from J1, J3, J4, J5, J6, and a combination thereof.
  • the one or more human VH gene segment is selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, a 6-1 human VH gene segment, and a combination thereof.
  • the mouse comprises a B cell that expresses a binding protein that specifically binds an antigen of interest, wherein the binding protein comprises a light chain derived from a human V ⁇ 1-39/J ⁇ 5 rearrangement or a human V ⁇ 3-20/J ⁇ 1 rearrangement, and wherein the cell comprises a rearranged immunoglobulin heavy chain gene derived from a rearrangement of human gene segments selected from a VH2-5, VH3-23, VH3-30, VH 4-39, VH4-59, and VH5-51 gene segment.
  • the one or more human VH gene segments are rearranged with a human heavy chain J gene segment selected from J1, J3, J4, J5, and J6.
  • the one or more human VH and J gene segments are rearranged with a human D gene segment selected from D1-7, D1-26, D3-3, D3-10, D3-16, D3-22, D5-5, D5-12, D6-6, D6-13, and D7-27.
  • the light chain gene has 1, 2, 3, 4, or 5 or more somatic hypermutations.
  • the mouse comprises a B cell that comprises a rearranged immunoglobulin heavy chain variable region gene sequence comprising a VH, JH, and DH gene segment selected from VH 2-5 + JH1 + D6-6, VH3-23 + JH4 + D3, VH3-23 + JH4 + D3-10, VH3-30 + JH1 + D6-6, VH3-30 +JH3 + D6-6, VH3-30 + JH4 + D1-7, VH3-30 + JH4 + D5-12, VH3-30 + JH4 + D6-13, VH3-30 + JH4 + D6-6, VH3-30 + JH4 + D7-27, VH3-30 + JH5 + D3-22, VH3-30 + JH5 + D6-6, VH3-30 + JH5 + D7-27, VH4-39 + JH3 + D1-26, VH4-59 + JH3 + D3-16, VH4-59 + JH3 + D3-22, VH4-59
  • the human VL gene segment is a human V ⁇ 1-39J ⁇ 5 gene segment
  • the mouse expresses a reverse chimeric light chain comprising (i) a VL domain derived from the human VL gene segment and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH and (ii) a somatically mutated human VH domain derived from a human VH gene segment selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and 6-1 human VH gene segment, and a combination thereof.
  • the mouse expresses a light chain that is somatically mutated.
  • the CL is a mouse ⁇ CL.
  • the human VL gene segment is a human V ⁇ 3-20J ⁇ 1 gene segment
  • the mouse expresses a reverse chimeric light chain comprising (i) a VL domain derived from the human VL gene segment, and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH, and (ii) a somatically mutated human VH derived from a human VH gene segment selected from a 1-2, 2-5, 3-7, 3-9, 3-11, 3-20, 3-23, 3-30, 3-33, 4-59, and 5-51 human VH gene segment, and a combination thereof.
  • the mouse expresses a light chain that is somatically mutated.
  • the CL is a mouse ⁇ CL.
  • a mouse comprising both a human V ⁇ 1-39J ⁇ 5 gene segment and a human V ⁇ 3-20J ⁇ 1 gene segment, and the mouse expresses a reverse chimeric light chain comprising (i) a VL domain derived from a human V ⁇ 1-39J ⁇ 5 gene segment or a human V ⁇ 3-20J ⁇ 1 gene segment, and (ii) a mouse CL; wherein the light chain is associated with a reverse chimeric heavy chain comprising (i) a mouse CH, and (ii) a somatically mutated human VH derived from a human VH gene segment selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, a 6-1 human VH gene segment, and a combination thereof.
  • the mouse expresses a light chain that is somatically mutated.
  • the CL is
  • the mouse comprises a replacement of 90-100% of the endogenous mouse heavy chain variable region gene locus with a plurality of human heavy chain variable region gene segments, wherein the human heavy chain variable region gene segments are operably linked to an endogenous mouse heavy chain constant gene, and the human heavy chain variable region gene segments are capable of rearranging and forming a rearranged human/mouse chimeric heavy chain gene.
  • all or substantially all of the endogenous unrearranged mouse VH gene segments are replaced with a plurality of unrearranged human VH gene segment.
  • the replacement is with at least 18, at least 39, at least 80, or 81 unrearranged human VH gene segments.
  • the replacement is with at least 12 functional unrearranged human VH gene segments, at least 25 functional unrearranged human VH gene segments, or at least 43 unrearranged human VH gene segments.
  • the genetically modified mouse is a C57BL strain, in a specific embodiment selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, C57BL/Ola.
  • the genetically modified mouse is a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain.
  • the mouse is a mix of aforementioned 129 strains, or a mix of aforementioned BL/6 strains.
  • the 129 strain of the mix is a 129S6 (129/SvEvTac) strain.
  • the mouse expresses a reverse chimeric antibody comprising a light chain that comprises a mouse ⁇ CL and a somatically mutated human VL domain derived from a human V ⁇ 1-39J ⁇ 5 gene segment or a human V ⁇ 3-20J ⁇ 1 gene segment, and a heavy chain that comprises a mouse CH and a somatically mutated human VH domain derived from a human VH gene segment selected from a 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and a 6-1 human VH gene segment, wherein the mouse does not express a fully mouse antibody and does not express a fully human antibody.
  • the mouse comprises a ⁇ light chain locus that comprises a replacement of endogenous mouse ⁇ VL gene segments with the human V ⁇ 1-39J ⁇ 5 gene segment or the human V ⁇ 3-20J ⁇ 1 gene segment, and comprises a replacement of all or substantially all endogenous mouse VH gene segments with a complete or substantially complete repertoire of human VH gene segments.
  • mouse cell that is isolated from a mouse as described herein.
  • a cell is provided where the cell is a cell isolated from a genetically modified mouse according to any one of the preceding claims, wherein the cell comprises:
  • hybridoma wherein the hybridoma is made with a B cell of a mouse as described herein.
  • the B cell is from a mouse as described herein that has been immunized with an immunogen comprising an epitope of interest, and the B cell expresses a binding protein that binds the epitope of interest, the binding protein has a somatically mutated human VH domain and a mouse CH, and has a human VL domain derived from a human V ⁇ 1-39J ⁇ 5 or a human V ⁇ 3-20J ⁇ 1 gene segment and a mouse CL.
  • mouse embryo wherein the embryo comprises a donor ES cell that is derived from a mouse as described herein.
  • a targeting vector comprising, from 5' to 3' in transcriptional direction with reference to the sequences of the 5' and 3' mouse homology arms of the vector, a 5' mouse homology arm, a human or mouse immunoglobulin promoter, a human or mouse leader sequence, and a human LCVR gene segment selected from a human V ⁇ 1-39J ⁇ 5 or a human V ⁇ 3-20J ⁇ 1 gene segment, and a 3' mouse homology arm.
  • the 5' and 3' homology arms target the vector to a sequence 5' with respect to an enhancer sequence that is present 5' and proximal to the mouse ⁇ constant region gene.
  • the promoter is a human immunoglobulin variable region gene segment promoter.
  • the promoter is a human V ⁇ 3-15 promoter.
  • the leader sequence is a mouse leader sequence.
  • the mouse leader sequence is a mouse V ⁇ 3-7 leader sequence.
  • a targeting vector is provided as described above, but in place of the 5' mouse homology arm the human or mouse promoter is flanked 5' with a site-specific recombinase recognition site (SRRS), and in place of the 3' mouse homology arm the human LCVR gene segment is flanked 3' with an SRRS.
  • SRRS site-specific recombinase recognition site
  • a reverse chimeric antibody made by a mouse as described herein, wherein the reverse chimeric antibody comprises a light chain comprising a mouse CL and a human VL, and a heavy chain comprising a human VH and a mouse CH.
  • Also provided is a method for making an antibody that binds an antigen of interest comprising immunizing the mouse provided with an antigen of interest, obtaining an immunoglobulin variable region gene sequence from the mouse and employing the immunoglobulin variable region gene sequence to produce an antibody that binds the antigen.
  • Also referred to is a method for making an antibody comprising expressing in a single cell (a) a first VH gene sequence of an immunized mouse as described herein fused with a human CH gene sequence; (b) a VL gene sequence of an immunized mouse as described herein fused with a human CL gene sequence; and, (c) maintaining the cell under conditions sufficient to express a fully human antibody, and isolating the antibody.
  • the cell comprises a second VH gene sequence of a second immunized mouse as described herein fused with a human CH gene sequence, the first VH gene sequence encodes a VH domain that recognizes a first epitope, and the second VH gene sequence encodes a VH domain that recognizes a second epitope, wherein the first epitope and the second epitope are not identical.
  • Also referred to is a method for making an epitope-binding protein comprising exposing a mouse as described herein with an immunogen that comprises an epitope of interest, maintaining the mouse under conditions sufficient for the mouse to generate an immunoglobulin molecule that specifically binds the epitope of interest, and isolating the immunoglobulin molecule that specifically binds the epitope of interest; wherein the epitope-binding protein comprises a heavy chain that comprises a somatically mutatedhuman VH and a mouse CH, associated with a light chain comprising a mouse CL and a human VL derived from a human V ⁇ 1-39 J ⁇ 5 or a human V ⁇ 3-20 J ⁇ 1 gene segment.
  • a cell that expresses an epitope-binding protein comprising: (a) a human VL nucleotide sequence encoding a human VL domain derived from a human V ⁇ 1-39J ⁇ 5 or a human V ⁇ 3-20J ⁇ 1 gene segment, wherein the human VL nucleotide sequence is fused (directly or through a linker) to a human immunoglobulin light chain constant domain cDNA sequence (e.g., a human ⁇ constant domain DNA sequence); and, (b) a first human VH nucleotide sequence encoding a human VH domain derived from a first human VH nucleotide sequence, wherein the first human VH nucleotide sequence is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain cDNA sequence; wherein the epitope-binding protein recognizes a first epitope.
  • the epitope-binding protein binds the first epitope with a dissociation constant of lower than 10 -6 M, lower than 10 -8 M, lower than 10 -9 M, lower than 10 -10 M, lower than 10 -11 M, or lower than 10 -12 M.
  • the cell comprises a second human VH nucleotide sequence encoding a second human VH domain, wherein the second human VH sequence is fused (directly or through a linker) to a human immunoglobulin heavy chain constant domain cDNA sequence, and wherein the second human VH domain does not specifically recognize the first epitope (e.g., displays a dissociation constant of, e.g., 10 -6 M, 10 -5 M, 10 -4 M, or higher), and wherein the epitope-binding protein recognizes the first epitope and the second epitope, and wherein the first and the second immunoglobulin heavy chains each associate with an identical light chain of (a).
  • the second human VH domain e.g., displays a dissociation constant of, e.g., 10 -6 M, 10 -5 M, 10 -4 M, or higher
  • the epitope-binding protein recognizes the first epitope and the second epitope, and wherein the first and the second immunoglobulin heavy
  • the second VH domain binds the second epitope with a dissociation constant that is lower than 10 -6 M, lower than 10 -7 M, lower than 10 -8 M, lower than 10 -9 M, lower than 10 -10 M, lower than 10 -11 M, or lower than 10 -12 M.
  • the epitope-binding protein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, each associated with an identical light chain derived from a human VL gene segment selected from a human V ⁇ 1-39J ⁇ 5 or a human V ⁇ 3-20J ⁇ 1 gene segment, wherein the first immunoglobulin heavy chain binds a first epitope with a dissociation constant in the nanomolar to picomolar range, the second immunoglobulin heavy chain binds a second epitope with a dissociation constant in the nanomolar to picomolar range, the first epitope and the second epitope are not identical, the first immunoglobulin heavy chain does not bind the second epitope or binds the second epitope with a dissociation constant weaker than the micromolar range (e.g., the millimolar range), the second immunoglobulin heavy chain does not bind the first epitope or binds the first epitope with a dissociation constant weaker than the micromolar range (
  • the first immunoglobulin heavy chain comprises a protein A-binding residue
  • the second immunoglobulin heavy chain lacks the protein A-binding residue
  • the cell is selected from CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6TM cell).
  • a reverse chimeric antibody is also referred to, comprising a human VH and a mouse heavy chain constant domain, a human VL and a mouse light chain constant domain, wherein the antibody is made by a process that comprises immunizing a mouse as described herein with an immunogen comprising an epitope, and the antibody specifically binds the epitope of the immunogen with which the mouse was immunized.
  • the VL domain is somatically mutated.
  • the VH domain is somatically mutated.
  • both the VL domain and the VH domain are somatically mutated.
  • the VL is linked to a mouse ⁇ constant domain.
  • the mouse comprises human heavy chain variable gene segments replacing all or substantially all mouse heavy chain variable gene segments at the endogenous mouse locus; no more than one human light chain variable gene segments selected from a rearranged V ⁇ 1-39/J and a rearranged V ⁇ 3-20/J segment, replacing all mouse light chain variable gene segments; wherein the human heavy chain variable gene segments are linked to an endogenous mouse heavy chain constant gene, and the human light chain variable gene segment is linked to a an endogenous mouse C ⁇ constant gene.
  • mouse ES cell comprising a replacement of all or substantially all mouse heavy chain variable gene segments with human heavy chain variable gene segments, and no more than one or two rearranged human light chain V/J segments, wherein the human heavy chain variable gene segments are linked to a mouse immunoglobulin heavy chain constant gene, and the human light chain V/J segments are linked to a mouse or human immunoglobulin light chain constant gene.
  • the light chain constant gene is a mouse constant gene.
  • an antigen-binding protein made by a mouse as described herein.
  • the antigen-binding protein comprises a human immunoglobulin heavy chain variable region fused with a mouse constant region, and a human immunoglobulin light chain variable region derived from a V ⁇ 1-39 gene segment or a V ⁇ 3-20 gene segment, wherein the light chain constant region is a mouse constant region.
  • a fully human antigen-binding protein made from an immunoglobulin variable region gene sequence from a mouse as described herein, wherein the antigen-binding protein comprises a fully human heavy chain comprising a human variable region derived from a sequence of a mouse as described herein, and a fully human light chain comprising a V ⁇ 1-39 or a V ⁇ 3-20 variable region.
  • the light chain variable region comprises one to five somatic mutations.
  • the light chain variable region is a cognate light chain variable region that is paired in a B cell of the mouse with the heavy chain variable region.
  • the fully human antigen-binding protein comprises a first heavy chain and a second heavy chain, wherein the first heavy chain and the second heavy chain comprise non-identical variable regions independently derived from a mouse as described herein, and wherein each of the first and second heavy chains express from a host cell associated with a human light chain derived from a V ⁇ 1-39 gene segment or a V ⁇ 3-20 gene segment.
  • the first heavy chain comprises a first heavy chain variable region that specifically binds a first epitope of a first antigen
  • the second heavy chain comprises a second heavy chain variable region that specifically binds a second epitope of a second antigen.
  • the first antigen and the second antigen are different.
  • the first antigen and the second antigen are the same, and the first epitope and the second epitope are not identical; in a specific instance, binding of the first epitope by a first molecule of the binding protein does not block binding of the second epitope by a second molecule of the binding protein.
  • a fully human binding protein derived from a human immunoglobulin sequence of a mouse as described herein comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, wherein the first immunoglobulin heavy chain comprises a first variable region that is not identical to a variable region of the second immunoglobulin heavy chain, and wherein the first immunoglobulin heavy chain comprises a wild-type protein A binding determinant, and the second heavy chain lacks a wild-type protein A binding determinant.
  • the first immunoglobulin heavy chain binds protein A under isolation conditions
  • the second immunoglobulin heavy chain does not bind protein A or binds protein A at least 10-fold, a hundred-fold, or a thousand-fold weaker than the first immunoglobulin heavy chain binds protein A under isolation conditions.
  • the first and the second heavy chains are IgG1 isotypes, wherein the second heavy chain comprises a modification selected from 95R (EU 435R), 96F (EU 436F), and a combination thereof, and wherein the first heavy chain lacks such modification.
  • a method is provided which is a method for making a human bispecific antibody, where the method comprises immunizing a mouse according to any one of claims 1 to 5 and making the bispecific antibody using human variable region gene sequences of B cells from the mouse.
  • the method for making a bispecific antigen-binding protein comprises exposing a first mouse as provided to a first antigen of interest that comprises a first epitope, exposing a second mouse as provided herein to a second antigen of interest that comprises a second epitope, allowing the first and the second mouseto each mount immune responses to the antigens of interest, identifying in the first mouse a first human heavy chain variable region that binds the first epitope of the first antigen of interest, identifying in the second mouse a second human heavy chain variable region that binds the second epitope of the second antigen of interest, making a first fully human heavy chain gene that encodes a first heavy chain that binds the first epitope of the first antigen of interest, making a second fully human heavy chain gene that encodes a second heavy chain that binds the second epitope of the second antigen of interest, expressing the first heavy chain and the second heavy chain in a cell that expresses a single fully human light chain derived from a human V
  • the first antigen and the second antigen are not identical.
  • first antigen and the second antigen are identical, and the first epitope and the second epitope are not identical. In one embodiment, binding of the first heavy chain variable region to the first epitope does not block binding of the second heavy chain variable region to the second epitope.
  • the first antigen is selected from a soluble antigen and a cell surface antigen (e.g., a tumor antigen), and the second antigen comprises a cell surface receptor.
  • the cell surface receptor is an immunoglobulin receptor.
  • the immunoglobulin receptor is an Fc receptor.
  • the first antigen and the second antigen are the same cell surface receptor, and binding of the first heavy chain to the first epitope does not block binding of the second heavy chain to the second epitope.
  • the light chain variable domain of the light chain comprises 2 to 5 somatic mutations. In one embodiment, the light chain variable domain is a somatically mutated cognate light chain expressed in a B cell of the first or the second immunized mouse with either the first or the second heavy chain variable domain.
  • the first fully human heavy chain bears an amino acid modification that reduces its affinity to protein A, and he second fully human heavy chain does not comprise a modification that reduces its affinity to protein A.
  • an antibody or a bispecific antibody which comprises a human heavy chain variable domain made in accordance with the invention.
  • use of a mouse provided to make a fully human antibody or a fully human bispecific antibody is provided.
  • antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable (VH) region and a heavy chain constant region (CH).
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable (VL) region and a light chain constant region (CL).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3.
  • the term "high affinity" antibody refers to an antibody that has a K D with respect to its target epitope about of 10 -9 M or lower (e.g., about 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or about 1 x 10 -12 M).
  • K D is measured by surface plasmon resonance, e.g., BIACORETM; in another embodiment, K D is measured by ELISA.
  • bispecific antibody includes an antibody capable of selectively binding two or more epitopes.
  • Bispecific antibodies generally comprise two nonidentical heavy chains, with each heavy chain specifically binding a different epitope-either on two different molecules (e.g., different epitopes on two different immunogens) or on the same molecule (e.g., different epitopes on the same immunogen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four or more orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa.
  • Epitopes specifically bound by the bispecific antibody can be on the same or a different target (e.g ., on the same or a different protein).
  • Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same immunogen.
  • nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same immunogen can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
  • a typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer epitope-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain epitope-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.
  • cell includes any cell that is suitable for expressing a recombinant nucleic acid sequence.
  • Cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E . coli, Bacillus spp., Streptomyces spp., etc .), mycobacteria cells, fungal cells, yeast cells ( e.g., S. cerevisiae, S. pombe, P. pastoris, P.
  • the cell is a human, monkey, ape, hamster, rat, or mouse cell.
  • the cell is eukaryotic and is selected from the following cells: CHO ( e.g ., CHO K1, DXB-11 CHO, Veggie-CHO), COS ( e.g ., COS-7), retinal cell, Vero, CV1, kidney ( e.g ., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, ( e.g ., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell.
  • the cell comprises one or more viral genes, e.g., a retinal
  • CDR complementarity determining region
  • a CDR includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes that normally ( i.e ., in a wild-type animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g ., an antibody or a T cell receptor).
  • a CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell.
  • a CDR can be somatically mutated (e.g ., vary from a sequence encoded in an animal's germline), humanized, and/or modified with amino acid substitutions, additions, or deletions.
  • CDRs can be encoded by two or more sequences (e.g. , germline sequences) that are not contiguous (e.g ., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g ., as the result of splicing or connecting the sequences (e.g ., V-D-J recombination to form a heavy chain CDR3).
  • conservative amino acid substitution when used to describe a conservative amino acid substitution, includes substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g ., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of a variable region to specifically bind a target epitope with a desired affinity.
  • groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine; acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine.
  • aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine
  • aliphatic-hydroxyl side chains such as serine and threonine
  • amide-containing side chains such as asparagine and glutamine
  • aromatic side chains such as phenylalanine, tyrosine, and trypto
  • Conservative amino acids substitution groups include, for example, valine/leucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine.
  • a conservative amino acid substitution can be substitution of any native residue in a protein with alanine, as used in, for example, alanine scanning mutagenesis.
  • a conservative substitution is made that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Exhaustive Matching of the Entire Protein Sequence Database, Science 256:1443-45 , hereby incorporated by reference.
  • the substitution is a moderately conservative substitution wherein the substitution has a nonnegative value in the PAM250 log-likelihood matrix.
  • residue positions in an immunoglobulin light chain or heavy chain differ by one or more conservative amino acid substitutions.
  • residue positions in an immunoglobulin light chain or functional fragment thereof e.g ., a fragment that allows expression and secretion from, e.g ., a B cell
  • residue positions in an immunoglobulin light chain or functional fragment thereof are not identical to a light chain whose amino acid sequence is listed herein, but differs by one or more conservative amino acid substitutions.
  • epitope-binding protein includes a protein having at least one CDR and that is capable of selectively recognizing an epitope, e.g ., is capable of binding an epitope with a K D that is at about one micromolar or lower (e.g ., a K D that is about 1 x 10 -6 M, 1 x 10 -7 M, 1 x 10 -9 M, 1 x 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or about 1 x 10 -12 M).
  • Therapeutic epitope-binding proteins e.g ., therapeutic antibodies
  • the phrase "functional fragment” includes fragments of epitope-binding proteins that can be expressed, secreted, and specifically bind to an epitope with a K D in the micromolar, nanomolar, or picomolar range. Specific recognition includes having a K D that is at least in the micromolar range, the nanomolar range, or the picomolar range.
  • germline includes reference to an immunoglobulin nucleic acid sequence in a non-somatically mutated cell, e.g ., a non-somatically mutated B cell or pre-B cell or hematopoietic cell.
  • heavy chain or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain constant region sequence from any organism.
  • Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof.
  • a typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain.
  • a functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an epitope (e.g ., recognizing the epitope with a K D in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.
  • an epitope e.g ., recognizing the epitope with a K D in the micromolar, nanomolar, or picomolar range
  • identity when used in connection with sequence, includes identity as determined by a number of different algorithms known in the art that can be used to measure nucleotide and/or amino acid sequence identity. In some embodiments described herein, identities are determined using a ClustalW v. 1.83 (slow) alignment employing an open gap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnet similarity matrix (MaeVectorTM 10.0.2, MacVector Inc., 2008).
  • the length of the sequences compared with respect to identity of sequences will depend upon the particular sequences, but in the case of a light chain constant domain, the length should contain sequence of sufficient length to fold into a light chain constant domain that is capable of self-association to form a canonical light chain constant domain, e.g ., capable of forming two beta sheets comprising beta strands and capable of interacting with at least one CH1 domain of a human or a mouse. In the case of a CH1 domain, the length of sequence should contain sequence of sufficient length to fold into a CH1 domain that is capable of forming two beta sheets comprising beta strands and capable of interacting with at least one light chain constant domain of a mouse or a human.
  • immunoglobulin molecule includes two immunoglobulin heavy chains and two immunoglobulin light chains.
  • the heavy chains may be identical or different, and the light chains may be identical or different.
  • light chain includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes human ⁇ and ⁇ light chains and a VpreB, as well as surrogate light chains.
  • Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified.
  • FR framework
  • a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain.
  • Light chains include those, e.g ., that do not selectively bind either a first or a second epitope selectively bound by the epitope-binding protein in which they appear.
  • Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more epitopes selectively bound by the epitope-binding protein in which they appear.
  • Common light chains are those derived from a human V ⁇ 1-39J ⁇ 5 gene segment or a human V ⁇ 3-20J ⁇ 1 gene segment, and include somatically mutated ( e.g ., affinity matured) versions of the same.
  • micromolar range is intended to mean 1-999 micromolar; the phrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase “picomolar range” is intended to mean 1-999 picomolar.
  • the phrase "somatically mutated” includes reference to a nucleic acid sequence from a B cell that has undergone class-switching, wherein the nucleic acid sequence of an immunoglobulin variable region (e.g ., a heavy chain variable domain or including a heavy chain CDR or FR sequence) in the class-switched B cell is not identical to the nucleic acid sequence in the B cell prior to class-switching, such as, for example, a difference in a CDR or framework nucleic acid sequence between a B cell that has not undergone class-switching and a B cell that has undergone class-switching.
  • an immunoglobulin variable region e.g ., a heavy chain variable domain or including a heavy chain CDR or FR sequence
  • “Somatically mutated” includes reference to nucleic acid sequences from affinity-matured B cells that are not identical to corresponding immunoglobulin variable region sequences in B cells that are not affinity-matured ( i.e., sequences in the genome of germline cells).
  • the phrase “somatically mutated” also includes reference to an immunoglobulin variable region nucleic acid sequence from a B cell after exposure of the B cell to an epitope of interest, wherein the nucleic acid sequence differs from the corresponding nucleic acid sequence prior to exposure of the B cell to the epitope of interest.
  • mutated refers to sequences from antibodies that have been generated in an animal, e.g ., a mouse having human immunoglobulin variable region nucleic acid sequences, in response to an immunogen challenge, and that result from the selection processes inherently operative in such an animal.
  • nucleic acid sequences that exist in the germline of an animal cell.
  • variable domain includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • mouse heavy chain immunoglobulin variable regions that pair with a common human light chain is of limited practical utility. More in vitro engineering efforts would be expended in a trial-and-error process to try to humanize the mouse heavy chain variable sequences while hoping to retain epitope specificity and affinity while maintaining the ability to couple with the common human light chain, with uncertain outcome. At the end of such a process, the final product may maintain some of the specificity and affinity, and associate with the common light chain, but ultimately immunogenicity in a human would likely remain a profound risk.
  • a suitable mouse for making human therapeutics would include a suitably large repertoire of human heavy chain variable region gene segments in place of endogenous mouse heavy chain variable region gene segments.
  • the human heavy chain variable region gene segments should be able to rearrange and recombine with an endogenous mouse heavy chain constant domain to form a reverse chimeric heavy chain (i.e., a heavy chain comprising a human variable domain and a mouse constant region).
  • the heavy chain should be capable of class switching and somatic hypermutation so that a suitably large repertoire of heavy chain variable domains are available for the mouse to select one that can associate with the limited repertoire of human light chain variable regions.
  • a mouse that selects a common light chain for a plurality of heavy chains has a practical utility.
  • antibodies that express in a mouse that can only express a common light chain will have heavy chains that can associate and express with an identical or substantially identical light chain. This is particularly useful in making bispecific antibodies.
  • a mouse can be immunized with a first immunogen to generate a B cell that expresses an antibody that specifically binds a first epitope.
  • the mouse (or a mouse genetically the same) can be immunized with a second immunogen to generate a B cell that expresses an antibody that specifically binds the second epitope.
  • Variable heavy regions can be cloned from the B cells and expresses with the same heavy chain constant region, and the same light chain, and expressed in a cell to make a bispecific antibody, wherein the light chain component of the bispecific antibody has been selected by a mouse to associate and express with the light chain component.
  • the inventors have engineered a mouse for generating immunoglobulin light chains that will suitably pair with a rather diverse family of heavy chains, including heavy chains whose variable regions depart from germline sequences, e.g ., affinity matured or somatically mutated variable regions.
  • the mouse is devised to pair human light chain variable domains with human heavy chain variable domains that comprise somatic mutations, thus enabling a route to high affinity binding proteins suitable for use as human therapeutics.
  • the genetically engineered mouse through the long and complex process of antibody selection within an organism, makes biologically appropriate choices in pairing a diverse collection of human heavy chain variable domains with a limited number of human light chain options.
  • the mouse is engineered to present a limited number of human light chain variable domain options in conjunction with a wide diversity of human heavy chain variable domain options.
  • the mouse Upon challenge with an immunogen, the mouse maximizes the number of solutions in its repertoire to develop an antibody to the immunogen, limited largely or solely by the number or light chain options in its repertoire. In various embodiments, this includes allowing the mouse to achieve suitable and compatible somatic mutations of the light chain variable domain that will nonetheless be compatible with a relatively large variety of human heavy chain variable domains, including in particular somatically mutated human heavy chain variable domains.
  • the mouse is engineered to render nonfunctional or substantially nonfunctional its ability to make, or rearrange, a native mouse light chain variable domain. This can be achieved, e.g ., by deleting the mouse's light chain variable region gene segments.
  • the endogenous mouse locus can then be modified by an exogenous suitable human light chain variable region gene segment of choice, operably linked to the endogenous mouse light chain constant domain, in a manner such that the exogenous human variable region gene segments can rearrange and recombine with the endogenous mouse light chain constant region gene and form a rearranged reverse chimeric light chain gene (human variable, mouse constant).
  • the light chain variable region is capable of being somatically mutated.
  • the appropriate enhancer(s) is retained in the mouse.
  • the mouse ⁇ intronic enhancer and mouse ⁇ 3' enhancer are functionally maintained, or undisrupted.
  • the genetically engineered mouse expresses a limited repertoire of reverse chimeric (human variable, mouse constant) light chains associated with a diversity of reverse chimeric (human variable, mouse constant) heavy chains.
  • the endogenous mouse ⁇ light chain variable region gene segments are deleted and replaced with a single human light chain variable region gene segments, operably linked to the endogenous mouse ⁇ constant region gene.
  • the mouse ⁇ intronic enhancer and the mouse ⁇ 3' enhancer are maintained.
  • the mouse also comprises a nonfunctional ⁇ light chain locus, or a deletion thereof or a deletion that renders the locus unable to make a ⁇ light chain.
  • mice comprising:
  • the genetically engineered mouse comprises a light chain variable region locus lacking an endogenous mouse light chain variable gene segment and comprising a human variable gene segment, in one embodiment a rearranged human V/J sequence, operably linked to a mouse constant region, wherein the locus is capable of undergoing somatic hypermutation, and wherein the locus expresses a light chain comprising the human V/J sequence linked to a mouse constant region.
  • the locus comprises a mouse ⁇ 3' enhancer, which is correlated with a normal, or wild-type, level of somatic hypermutation.
  • the genetically engineered mouse in various embodiments when immunized with an antigen of interest generates B cells that exhibit a diversity of rearrangements of human immunoglobulin heavy chain variable regions that express and function with one rearranged light chain, including embodiments where the one light chains comprise human light chain variable regions that comprise, e.g., 1 to 5 somatic mutations.
  • the human light chains so expressed are capable of associating and expressing with any human immunoglobulin heavy chain variable region expressed in the mouse.
  • compositions and methods of described herein can be used to make binding proteins that bind more than one epitope with high affinity, e.g., bispecific antibodies.
  • Advantages of the invention include the ability to select suitably high binding (e.g., affinity matured) heavy chain immunoglobulin chains each of which will associate with a single light chain.
  • each construct encodes a human heavy chain variable domain that binds a different epitope.
  • One of the human VLs e.g., human V ⁇ 1-39J ⁇ 5 or human V ⁇ 3-20J ⁇ 1
  • a suitable human constant region gene e.g., a human ⁇ constant gene.
  • one of the heavy chains is modified to omit a Protein A-binding determinant, resulting in a differential affinity of a homodimeric binding protein from a heterodimeric binding protein.
  • Compositions and methods that address this issue are described in USSN 12/832,838, filed 25 June 20010 , entitled “Readily Isolated Bispecific Antibodies with Native Immunoglobulin Format," published as US 2010/0331527A1 .
  • an epitope-binding protein as described herein is referred to, wherein human VL and VH sequences are derived from mice described herein that have been immunized with an antigen comprising an epitope of interest.
  • an epitope-binding protein that comprises a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first epitope-binding region that selectively binds a first epitope, followed by a constant region that comprises a first CH3 region of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and, a second polypeptide comprising, from N-terminal to C-terminal, a second epitope-binding region that selectively binds a second epitope, followed by a constant region that comprises a second CH3 region of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to protein A.
  • the second CH3 region comprises an H95R modification (by IMGT exon numbering; H435R by EU numbering). In another embodiment, the second CH3 region further comprises a Y96F modification (IMGT; Y436F by EU).
  • the second CH3 region is from a modified human IgG1, and further comprises a modification selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU).
  • the second CH3 region is from a modified human IgG2, and further comprises a modification selected from the group consisting of N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU).
  • IMGT N44S, K52N, and V82I
  • the second CH3 region is from a modified human IgG4, and further comprises a modification selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU).
  • IMGT Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I
  • One method for making an epitope-binding protein that binds more than one epitope is to immunize a first mouse in accordance with the invention with an antigen that comprises a first epitope of interest, wherein the mouse comprises an endogenous immunoglobulin light chain variable region locus that does not contain an endogenous mouse VL that is capable of rearranging and forming a light chain, wherein at the endogenous mouse immunglobulin light chain variable region locus is a single human VL gene segment operably linked to the mouse endogenous light chain constant region gene, and the human VL gene segment is selected from a human V ⁇ 1-39J ⁇ 5 and a human V ⁇ 3-20J ⁇ 1, and the endogenous mouse VH gene segments have been replaced in whole or in part with human VH gene segments, such that immunoglobulin heavy chains made by the mouse are solely or substantially heavy chains that comprise human variable domains and mouse constant domains.
  • a reverse chimeric antibody comprising only one of two human light chain variable domains (e.g., one of human V ⁇ 1-39J ⁇ 5 or human V ⁇ 3-20J ⁇ 1).
  • the nucleotide sequence of the VH (and, optionally, the VL) can be retrieved (e.g., by PCR) and cloned into an expression construct in frame with a suitable human immunoglobulin constant domain.
  • This process can be repeated to identify a second VH domain that binds a second epitope, and a second VH gene sequence can be retrieved and cloned into an expression vector in frame to a second suitable immunoglobulin constant domain.
  • the first and the second immunoglobulin constant domains can the same or different isotype, and one of the immunoglobulin constant domains (but not the other) can be modified as described herein or in US 2010/0331527A1 , and epitope-binding protein can be expressed in a suitable cell and isolated based on its differential affinity for Protein A as compared to a homodimeric epitope-binding protein, e.g., as described in US 2010/0331527A1 .
  • a method for making a bispecific epitope-binding protein comprising identifying a first affinity-matured (e.g., comprising one or more somatic hypermutations) human VH nucleotide sequence (VH1) from a mouse as described herein, identifying a second affinity-matured (e.g., comprising one or more somatic hypermutations) human VH nucleotide sequence (VH2) from a mouse as described herein, cloning VH1 in frame with a human heavy chain lacking a Protein A-determinant modification as described in US 2010/0331527A1 for form heavy chain 1 (HC1), cloning VH2 in frame with a human heavy chain comprising a Protein A-determinant as described in US 2010/0331527A1 to form heavy chain 2 (HC2), introducing an expression vector comprising HC1 and the same or a different expression vector comprising HC2 into a cell, wherein the cell also expresses a human immunoglobul
  • HC1 is an IgG1
  • HC2 is an IgG1 that comprises the modification H95R (IMGT; H435R by EU) and further comprises the modification Y96F (IMGT; Y436F by EU).
  • the VH domain encoded by VH1, the VH domain encoded by VH2, or both, are somatically mutated.
  • a variety of human variable regions from affinity-matured antibodies raised against four different antigens were expressed with either their cognate light chain, or at least one of a human light chain selected from human V ⁇ 1-39J ⁇ 5, human V ⁇ 3-20J ⁇ 1, or human VpreBJ ⁇ 5 (see Example 1).
  • somatically mutated high affinity heavy chains from different gene families paired successfully with rearranged human germline V ⁇ 1-39J ⁇ 5 and V ⁇ 3-20J ⁇ 1 regions and were secreted from cells expressing the heavy and light chains.
  • VH domains derived from the following human VH families expressed favorably: 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51, and 6-1.
  • a mouse that is engineered to express a limited repertoire of human VL domains from one or both of V ⁇ 1-39J ⁇ 5 and V ⁇ 3-20J ⁇ 1 will generate a diverse population of somatically mutated human VH domains from a VH locus modified to replace mouse VH gene segments with human VH gene segments.
  • Mice genetically engineered to express reverse chimeric (human variable, mouse constant) immunoglobulin heavy chains associated with a single rearranged light chain e.g ., a V ⁇ 1-39/J or a V ⁇ 3-20/J
  • a single rearranged light chain e.g ., a V ⁇ 1-39/J or a V ⁇ 3-20/J
  • B cells that comprised a diversity of human V segment rearrangements and expressed a diversity of high-affinity antigen-specific antibodies with diverse properties with respect to their ability to block binding of the antigen to its ligand, and with respect to their ability to bind variants of the antigen (see Examples 5 through 10).
  • mice and methods described herein are useful in making and selecting human immunoglobulin heavy chain variable domains, including somatically mutated human heavy chain variable domains, that result from a diversity of rearrangements, that exhibit a wide variety of affinities (including exhibiting a K D of about a nanomolar or less), a wide variety of specificities (including binding to different epitopes of the same antigen), and that associate and express with the same or substantially the same human immunoglobulin light chain variable region.
  • Example 1 Identification of human heavy chain variable regions that associate with selected human light chain variable regions
  • An in vitro expression system was constructed to determine if a single rearranged human germline light chain could be co-expressed with human heavy chains from antigen specific human antibodies.
  • VELOCIMMUNE® Methods for generating human antibodies in genetically modified mice are known (see e.g ., US 6,596,541 , Regeneron Pharmaceuticals, VELOCIMMUNE®).
  • the VELOCIMMUNE® technology involves generation of a genetically modified mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation.
  • the DNA encoding the variable regions of the heavy and light chains of the antibodies produced from a VELOCIMMUNE® mouse are fully human. Initially, high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
  • the mouse constant regions are replaced with a desired human constant region to generate a fully human antibody containing a non-IgM isotype, for example, wild-type or modified IgG1, IgG2, IgG3 or IgG4. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
  • a VELOCIMMUNE® mouse was immunized with a growth factor that promotes angiogenesis (Antigen C) and antigen-specific human antibodies were isolated and sequenced for V gene usage using standard techniques recognized in the art. Selected antibodies were cloned onto human heavy and light chain constant regions and 69 heavy chains were selected for pairing with one of three human light chains: (1) the cognate ⁇ light chain linked to a human ⁇ constant region, (2) a rearranged human germline V ⁇ 1-39J ⁇ 5 linked to a human ⁇ constant region, or (3) a rearranged human germline V ⁇ 3-20J ⁇ 1 linked to a human ⁇ constant region. Each heavy chain and light chain pair were co-transfected in CHO-K1 cells using standard techniques.
  • Presence of antibody in the supernatant was detected by anti-human IgG in an ELISA assay.
  • Antibody titer (ng/ml) was determined for each heavy chain/light chain pair and titers with the different rearranged germline light chains were compared to the titers obtained with the parental antibody molecule ( i.e., heavy chain paired with cognate light chain) and percent of native titer was calculated (Table 1).
  • V H Heavy chain variable gene.
  • ND no expression detected under current experimental conditions.
  • VELOCIMMUNE® mice were immunized with several different antigens and selected heavy chains of antigen specific human antibodies were tested for their ability to pair with different rearranged human germline light chains (as described above).
  • the antigens used in this experiment included an enzyme involved in cholesterol homeostasis (Antigen A), a serum hormone involved in regulating glucose homeostasis (Antigen B), a growth factor that promotes angiogenesis (Antigen C) and a cell-surface receptor (Antigen D).
  • Antigen specific antibodies were isolated from mice of each immunization group and the heavy chain and light chain variable regions were cloned and sequenced.
  • V gene usage was determined and selected heavy chains were paired with either their cognate light chain or a rearranged human germline V ⁇ 1-39J ⁇ 5 region.
  • Each heavy/light chain pair was co-transfected in CHO-K1 cells and the presence of antibody in the supernatant was detected by anti-human IgG in an ELISA assay.
  • Antibody titer ( ⁇ g/ml) was determined for each heavy chain/light chain pairing and titers with the different rearranged human germline light chains were compared to the titers obtained with the parental antibody molecule (i.e., heavy chain paired with cognate light chain) and percent of native titer was calculated (Table 2).
  • V H Heavy chain variable gene.
  • V ⁇ ⁇ light chain variable gene. ND: no expression detected under current experimental conditions.
  • both rearranged human germline light chains conferred an increase in expression as compared to the cognate light chain of the parental antibody.
  • the rearranged human germline V ⁇ 1-39J ⁇ 5 region conferred an increase in expression of several heavy chains specific for a range of different classes of antigens as compared to the cognate light chain for the parental antibodies.
  • Antibody titer was increased by more than two-fold for about 35% (15/43) of the heavy chains as compared to the cognate light chain of the parental antibodies. For two heavy chains (315 and 316), the increase was greater than ten-fold as compared to the parental antibody.
  • V H 3 family three heavy chains are over represented in comparison to other heavy chain variable region gene families. This demonstrates a favorable relationship of human V H 3 heavy chains to pair with rearranged human germline V ⁇ 1-39J ⁇ 5 and V ⁇ 3-20J ⁇ 1 light chains.
  • the human variable gene segments used for constructing these three regions included rearranged human V ⁇ 1-39J ⁇ 5 sequence, a rearranged human V ⁇ 3-20J ⁇ 1 sequence and a rearranged human VpreBJ ⁇ 5 sequence.
  • a DNA segment containing exon 1 (encoding the leader peptide) and intron 1 of the mouse V ⁇ 3-7 gene was made by de novo DNA synthesis (Integrated DNA Technologies). Part of the 5' untranslated region up to a naturally occurring Blpl restriction enzyme site was included. Exons of human V ⁇ 1-39 and V ⁇ 3-20 genes were PCR amplified from human genomic BAC libraries. The forward primers had a 5' extension containing the splice acceptor site of intron 1 of the mouse V ⁇ 3-7 gene.
  • the reverse primer used for PCR of the human V ⁇ 1-39 sequence included an extension encoding human J ⁇ 5, whereas the reverse primer used for PCR of the human V ⁇ 3-20 sequence included an extension encoding human J ⁇ 1.
  • the human VpreBJ ⁇ 5 sequence was made by de novo DNA synthesis (Integrated DNA Technologies). A portion of the human J ⁇ -C ⁇ intron including the splice donor site was PCR amplified from plasmid pBS-296-HA18-PIScel. The forward PCR primer included an extension encoding part of either a human J ⁇ 5, J ⁇ 1, or J ⁇ 5 sequence. The reverse primer included a PI-Scel site, which was previously engineered into the intron.
  • mice V ⁇ 3-7 exon1/intron 1, human variable light chain exons, and human J ⁇ -C ⁇ intron fragments were sewn together by overlap extension PCR, digested with Blpl and PI-Scel, and ligated into plasmid pBS-296-HA18-PIScel, which contained the promoter from the human V ⁇ 3-15 variable gene segment.
  • a loxed hygromycin cassette within plasmid pBS-296-HA18-PIScel was replaced with a FRTed hygromycin cassette flanked by NotI and AscI sites.
  • the NotI/PI-SceI fragment of this plasmid was ligated into modified mouse BAC 254m04, which contained part of the mouse J ⁇ -C ⁇ intron, the mouse C ⁇ exon, and about 75 kb of genomic sequence downstream of the mouse ⁇ locus which provided a 3' homology arm for homologous recombination in mouse ES cells.
  • the Notl/Ascl fragment of this BAC was then ligated into modified mouse BAC 302g12, which contained a FRTed neomycin cassette and about 23 kb of genomic sequence upstream of the endogenous ⁇ locus for homologous recombination in mouse ES cells.
  • Restriction enzyme sites were introduced at the 5' and 3' ends of an engineered light chain insert for cloning into a targeting vector: an Ascl site at the 5' end and a PI-Scel site at the 3' end.
  • the targeting construct from 5' to 3' included a 5' homology arm containing sequence 5' to the endogenous mouse ⁇ light chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance gene, an genomic sequence including the human V ⁇ 3-15 promoter, a leader sequence of the mouse V ⁇ 3-7 variable gene segment, a intron sequence of the mouse V ⁇ 3-7 variable gene segment, an open reading frame of a rearranged human germline V ⁇ 1-39J ⁇ 5 region, a genomic sequence containing a portion of the human J ⁇ -C ⁇ intron, and a 3' homology arm containing sequence 3' of the endogenous mouse J ⁇ 5 gene segment obtained from mouse BAC clo
  • Targeted insertion of the rearranged human germline V ⁇ 1-39J ⁇ 5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germline light chain region. Briefly, the intron sequence 3' to the mouse V ⁇ 3-7 leader sequence was confirmed with primers ULC-m1F (AGGTGAGGGT ACAGATAAGT GTTATGAG; SEQ ID NO:2) and ULC-m1R (TGACAAATGC CCTAATTATA GTGATCA; SEQ ID NO:3).
  • the open reading frame of the rearranged human germline V ⁇ 1-39J ⁇ 5 region was confirmed with primers 1633-h2F (GGGCAAGTCA GAGCATTAGC A; SEQ ID NO:4) and 1633-h2R (TGCAAACTGG ATGCAGCATA G; SEQ ID NO:5).
  • the neomycin cassette was confirmed with primers neoF (GGTGGAGAGG CTATTCGGC; SEQ ID NO:6) and neoR (GAACACGGCG GCATCAG; SEQ ID NO:7).
  • Targeted BAC DNA was then used to electroporate mouse ES cells to created modified ES cells for generating chimeric mice that express a rearranged human germline V ⁇ 1-39J ⁇ 5 region.
  • Positive ES cell clones were confirmed by TAQMANTM screening and karyotyping using probes specific for the engineered V ⁇ 1-39J ⁇ 5 light chain region inserted into the endogenous locus. Briefly, probe neoP (TGGGCACAAC AGACAATCGG CTG; SEQ ID NO:8) which binds within the neomycin marker gene, probe ULC-m1 P (CCATTATGAT GCTCCATGCC TCTCTGTTC; SEQ ID NO:9) which binds within the intron sequence 3' to the mouse V ⁇ 3-7 leader sequence, and probe 1633h2P (ATCAGCAGAA ACCAGGGAAA GCCCCT; SEQ ID NO:10) which binds within the rearranged human germline V ⁇ 1-39J ⁇ 5 open reading frame. Positive ES cell clones were then used to implant female mice to give rise to a litter of pups expressing the germline V ⁇ 1-39J ⁇ 5 light chain region.
  • ES cells bearing the rearranged human germline V ⁇ 1-39J ⁇ 5 light chain region are transfected with a constuct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting construct.
  • the neomycin cassette is removed by breeding to mice that express FLP recombinase ( e.g ., US 6,774,279 ).
  • the neomycin cassette is retained in the mice
  • an engineered light chain locus expressing a rearranged human germline V ⁇ 3-20J ⁇ 1 region was made using a targeting construct including, from 5' to 3', a 5' homology arm containing sequence 5' to the endogenous mouse ⁇ light chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance gene, a genomic sequence including the human V ⁇ 3-15 promoter, a leader sequence of the mouse V ⁇ 3-7 variable gene segment, an intron sequence of the mouse V ⁇ 3-7 variable gene segment, an open reading frame of a rearranged human germline V ⁇ 3-20J ⁇ 1 region, a genomic sequence containing a portion of the human J ⁇ -C ⁇ intron, and a 3' homology arm containing sequence 3' of the endogenous mouse J ⁇ 5 gene segment obtained from mouse BAC clone 254m04 ( Figure 2 , middle).
  • the sequence of the engineered human V ⁇ 3-20J ⁇ 1 locus is shown in SEQ ID NO:11.
  • Targeted insertion of the rearranged human germline V ⁇ 3-20J ⁇ 1 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germline V ⁇ 3-20J ⁇ 1 light chain region.
  • PCR polymerase chain reaction
  • the intron sequence 3' to the mouse V ⁇ 3-7 leader sequence was confirmed with primers ULC-m1 F (SEQ ID NO:2) and ULC-m1 R (SEQ ID NO:3).
  • the open reading frame of the rearranged human germline V ⁇ 3-20J ⁇ 1 region was confirmed with primers 1635-h2F (TCCAGGCACC CTGTCTTTG; SEQ ID NO:12) and 1635-h2R (AAGTAGCTGC TGCTAACACT CTGACT; SEQ ID NO:13).
  • neomycin cassette was confirmed with primers neoF (SEQ ID NO:6) and neoR (SEQ ID NO:7).
  • Targeted BAC DNA was then used to electroporate mouse ES cells to created modified ES cells for generating chimeric mice that express the rearranged human germline V ⁇ 3-20J ⁇ 1 light chain.
  • Positive ES cell clones were confirmed by TaqmanTM screening and karyotyping using probes specific for the engineered V ⁇ 3-20J ⁇ 1 light chain region inserted into the endogenous ⁇ light chain locus. Briefly, probe neoP (SEQ ID NO:8) which binds within the neomycin marker gene, probe ULC-m1 P (SEQ ID NO:9) which binds within the mouse V ⁇ 3-7 leader sequence, and probe 1635h2P (AAAGAGCCAC CCTCTCCTGC AGGG; SEQ ID NO:14) which binds within the human V ⁇ 3-20J ⁇ 1 open reading frame. Positive ES cell clones were then used to implant female mice. A litter of pups expressing the human germline V ⁇ 3-20J ⁇ 1 light chain region.
  • ES cells bearing human germline V ⁇ 3-20J ⁇ 1 light chain region can be transfected with a constuct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting consruct.
  • the neomycin cassette may be removed by breeding to mice that express FLP recombinase ( e.g ., US 6,774,279 ).
  • the neomycin cassette is retained in the mice.
  • an engineered light chain locus expressing a rearranged human germline VpreBJ ⁇ 5 region was made using a targeting construct including, from 5' to 3', a 5' homology arm containing sequence 5' to the endogenous mouse ⁇ light chain locus obtained from mouse BAC clone 302g12, a FRTed neomycin resistance gene, an genomic sequence including the human V ⁇ 3-15 promoter, a leader sequence of the mouse V ⁇ 3-7 variable gene segment, an intron sequence of the mouse V ⁇ 3-7 variable gene segment, an open reading frame of a rearranged human germline VpreBJ ⁇ 5 region, a genomic sequence containing a portion of the human J ⁇ -C ⁇ intron, and a 3' homology arm containing sequence 3' of the endogenous mouse J ⁇ 5 gene segment obtained from mouse BAC clone 254m04
  • Targeted insertion of the rearranged human germline VpreBJ ⁇ 5 region into BAC DNA was confirmed by polymerase chain reaction (PCR) using primers located at sequences within the rearranged human germline VpreBJ ⁇ 5 region light chain region.
  • PCR polymerase chain reaction
  • the intron sequence 3' to the mouse V ⁇ 3-7 leader sequence was confirmed with primers ULC-m1F (SEQ ID NO:2) and ULC-m1R (SEQ ID NO:3).
  • the open reading frame of the rearranged human germline VpreBJ ⁇ 5 region was confirmed with primers 1616-h1F (TGTCCTCGGC CCTTGGA; SEQ ID NO:16) and 1616-h1R (CCGATGTCAT GGTCGTTCCT; SEQ ID NO:17).
  • neomycin cassette was confirmed with primers neoF (SEQ ID NO:6) and neoR (SEQ ID NO:7).
  • Targeted BAC DNA was then used to electroporate mouse ES cells to created modified ES cells for generating chimeric mice that express the rearranged human germline VpreBJ ⁇ 5 light chain.
  • Positive ES cell clones are confirmed by TAQMANTM screening and karyotyping using probes specific for the engineered VpreBJ ⁇ 5 light chain region inserted into the endogenous ⁇ light chain locus. Briefly, probe neoP (SEQ ID NO:8) which binds within the neomycin marker gene, probe ULC-m1 P (SEQ ID NO:9) which binds within the mouse IgV ⁇ 3-7 leader sequence, and probe 1616h1P (ACAATCCGCC TCACCTGCAC CCT; SEQ ID NO:18) which binds within the human VpreBJ ⁇ 5 open reading frame. Positive ES cell clones are then used to implant female mice to give rise to a litter of pups expressing a germline light chain region.
  • ES cells bearing the rearranged human germline VpreBJ ⁇ 5 light chain region are transfected with a construct that expresses FLP in order to remove the FRTed neomycin cassette introduced by the targeting consruct.
  • the neomycin cassette is removed by breeding to mice that express FLP recombinase (e.g., US 6,774,279 ).
  • the neomycin cassette is retained in the mice.
  • Targeted ES cells described above were used as donor ES cells and introduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method (see, e.g., US Pat. No. 7,294,754 and Poueymirou et al. (2007) F0 generation mice that are essentially fully derived from the donor gene-targeted ES cells allowing immediate phenotypic analyses Nature Biotech. 25(1):91-99 .
  • VELOCIMICE® independently bearing an engineered human germline V ⁇ 1-39J ⁇ 5 light chain region, a V ⁇ 3-20J ⁇ 1 light chain region or a VpreBJ ⁇ 5 light chain region are identified by genotyping using a modification of allele assay (Valenzuela et al., supra) that detects the presence of the unique rearranged human germline light chain region.
  • Pups are genotyped and a pup heterozygous for the unique rearranged human germline light chain region are selected for characterizing expression of the rearranged human germline light chain region.
  • mice bearing one of the rearranged human germline light chain regions are bred to another mouse containing a deletion in the endogenous ⁇ light chain locus.
  • the progeny obtained will express, as their only light chain, the rearranged human germline light chain region as described in Example 2. Breeding is performed by standard techniques recognized in the art and, alternatively, by a commercial breeder (e.g., The Jackson Laboratory).
  • Mouse strains bearing an engineered light chain locus and a deletion of the endogenous ⁇ light chain locus are screened for presence of the unique light chain region and absence of endogenous mouse ⁇ light chains.
  • mice bearing an engineered human germline light chain locus are bred with mice that contain a replacement of the endogenous mouse heavy chain variable gene locus with the human heavy chain variable gene locus (see US 6,596,541 ; the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.).
  • the VELOCIMMUNE® mouse comprises a genome comprising human heavy chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies comprising a human heavy chain variable region and a mouse heavy chain constant region in response to antigenic stimulation.
  • the DNA encoding the variable regions of the heavy chains of the antibodies is isolated and operably linked to DNA encoding the human heavy chain constant regions.
  • the DNA is then expressed in a cell capable of expressing the fully human heavy chain of the antibody.
  • mice bearing a replacement of the endogenous mouse VH locus with the human VH locus and a single rearranged human germline VL region at the endogenous ⁇ light chain locus are obtained.
  • Reverse chimeric antibodies containing somatically mutated heavy chains (human VH and mouse CH) with a single human light chain (human VL and mouse CL) are obtained upon immunization with an antigen of interest.
  • VH and VL nucleotide sequences of B cells expressing the antibodies are identified and fully human antibodies are made by fusion the VH and VL nucleotide sequences to human CH and CL nucleotide sequences in a suitable expression system.
  • Example 5 Generation of Antibodies from Mice Expressing Human Heavy Chains and a Rearranged Human Germline Light Chain Region
  • mice After breeding mice that contain the engineered human light chain region to various desired strains containing modifications and deletions of other endogenous Ig loci (as described in Example 4), selected mice can be immunized with an antigen of interest.
  • a VELOCIMMUNE® mouse containing one of the single rearranged human germline light chain regions is challenged with an antigen, and lymphatic cells (such as B-cells) are recovered from serum of the animals.
  • lymphatic cells such as B-cells
  • the lymphatic cells are fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies containing human heavy chain variables and a rearranged human germline light chains which are specific to the antigen used for immunization.
  • DNA encoding the variable regions of the heavy chains and the light chain are isolated and linked to desirable isotypic constant regions of the heavy chain and light chain.
  • the single light chain of each antibody may be somatically mutated. This adds additional diversity to the antigen-specific repertoire comprising a single light chain and diverse heavy chain sequences.
  • the resulting cloned antibody sequences are subsequently expressed in a cell, such as a CHO cell.
  • DNA encoding the antigen-specific chimeric antibodies or the variable domains of the light and heavy chains are identified directly from antigen-specific lymphocytes.
  • high affinity chimeric antibodies are isolated having a human variable region and a mouse constant region.
  • the antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc.
  • the mouse constant regions are replaced with a desired human constant region to generate the fully human antibody containing a somatically mutated human heavy chain and a single light chain derived from a rearranged human germline light chain region of the invention.
  • Suitable human constant regions include, for example wild-type or modified IgG1 or IgG4.
  • Antigen E Two to three micrograms of Antigen E are mixed with 10 ⁇ g of CpG oligonucleotide (Cat # tlrl-modn - ODN1826 oligonucleotide ; InVivogen, San Diego, CA) and 25 ⁇ g of Adju-Phos (Aluminum phosphate gel adjuvant, Cat# H-71639-250; Brenntag Biosector, Frederikssund, Denmark) prior to injection. A total of six injections are given prior to the final antigen recall, which is given 3-5 days prior to sacrifice. Bleeds after the 4th and 6th injection are collected and the antibody immune response is monitored by a standard antigen-specific immunoassay.
  • CpG oligonucleotide Cat # tlrl-modn - ODN1826 oligonucleotide ; InVivogen, San Diego, CA
  • Adju-Phos Alluminum phosphate gel adjuvant, Cat# H-71639-
  • splenocytes are harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines.
  • the hybridoma cell lines are screened and selected to identify cell lines that produce Antigen E-specific common light chain antibodies.
  • anti-Antigen E-specific common light chain antibodies i.e., antibodies possessing human heavy chain variable domains, the same human light chain variable domain, and mouse constant domains.
  • anti-Antigen E common light chain antibodies are isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in U.S. 2007/0280945A1 .
  • several fully human anti-Antigen E common light chain antibodies i.e. , antibodies possessing human heavy chain variable domains, either an engineered human V ⁇ 1-39J ⁇ 5 light chain or an engineered human V ⁇ 3-20J ⁇ 1 light chain region, and human constant domains
  • nucleic acids encoding heavy chain antibody variable regions were cloned and sequenced. From the nucleic acid sequences and predicted amino acid sequences of the antibodies, gene usage was identified for the heavy chain variable region (HCVR) of selected common light chain antibodies obtained from immunized VELOCIMMUNE® mice containing either the engineered human V ⁇ 1-39J ⁇ 5 light chain or engineered human V ⁇ 3-20J ⁇ 1 light chain region.
  • HCVR heavy chain variable region
  • mice according to the invention generate antigen-specific common light chain antibodies from a variety of human heavy chain gene segments, due to a variety of rearrangements, when employing either a mouse that expresses a light chain from only a human V ⁇ 1-39- or a human V ⁇ 3-20-derived light chain.
  • Human V H gene segments of the 2, 3, 4, and 5 families rearranged with a variety of human D H segments and human J H segments to yield antigen-specific antibodies.
  • the extracellular domain (ECD) of Antigen E was conjugated to two myc epitope tags and a 6X histidine tag (Antigen E-mmH) and amine-coupled to carboxylated microspheres at a concentration of 20 ⁇ g/mL in MES buffer. The mixture was incubated for two hours at room temperature followed by bead deactivation with 1 M Tris pH 8.0 followed by washing in PBS with 0.05% (v/v) Tween-20. The beads were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO).
  • Detection of biotinylated-Ligand Y bound to Antigen E-myc-myc-6His labeled beads was determined with R-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, MD) followed by measurement in a LuminexTM flow cytometry-based analyzer. Background Mean Fluorescence Intensity (MFI) of a sample without Ligand Y was subtracted from all samples. Percent blocking was calculated by division of the background-subtracted MFI of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.
  • MFI Green Fluorescence Intensity
  • Ligand Y was amine-coupled to carboxylated microspheres at a concentration of 20 ⁇ g/mL diluted in MES buffer. The mixture and incubated two hours at room temperature followed by deactivation of beads with 1 M Tris pH 8 then washing in PBS with 0.05% (v/v) Tween-20. The beads were then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 2% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO). In a 96-well filter plate, supernatants containing Antigen E-specific common light chain antibodies were diluted 1:15 in buffer. A negative control containing a mock supernatant with the same media components as for the antibody supernatant was prepared.
  • PBS Irvine Scientific, Santa Ana, CA
  • BSA Sigma-Aldrich Corp., St. Louis, MO
  • a biotinylated-Antigen E-mmH was added to a final concentration of 0.42 nM and incubated overnight at 4°C.
  • Ligand Y-labeled beads were then added to the antibody/Antigen E mixture and incubated for two hours at room temperature.
  • Detection of biotinylated-Antigen E-mmH bound to Ligand Y-beads was determined with R-Phycoerythrin conjugated to Streptavidin (Moss Inc, Pasadena, MD) followed by measurement in a LuminexTM flow cytometry-based analyzer. Background Mean Fluorescence Intensity (MFI) of a sample without Antigen E was subtracted from all samples. Percent blocking was calculated by division of the background-subtracted MFI of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.
  • MFI Mean Fluorescence Intensity
  • Tables 5 and 6 show the percent blocking for all 98 anti-Antigen E common light chain antibodies tested in both LuminexTM assays. ND: not determined under current experimental conditions.
  • the same 80 common light chain antibodies containing the V ⁇ 1-39J ⁇ 5 engineered light chain were tested for their ability to block binding of Antigen E to Ligand Y-labeled beads.
  • 36 demonstrated >50% blocking, while 44 demonstrated ⁇ 50% blocking (27 at 25-50% blocking and 17 at ⁇ 25% blocking).
  • 18 common light chain antibodies containing the V ⁇ 3-20J ⁇ 1 engineered light chain 1 demonstrated >50% blocking, while 17 demonstrated ⁇ 50% blocking (5 at 25-50% blocking and 12 at ⁇ 25% blocking) of Antigen E binding to Ligand Y-labeled beads.
  • Tables 5 and 6 establish that the rearrangements described in Tables 3 and 4 generated anti-Antigen E-specific common light chain antibodies that blocked binding of Ligand Y to its cognate receptor Antigen E with varying degrees of efficacy, which is consistent with the anti-Antigen E common light chain antibodies of Tables 3 and 4 comprising antibodies with overlapping and non-overlapping epitope specificity with respect to Antigen E.
  • Human common light chain antibodies raised against Antigen E were tested for their ability to block Antigen E binding to a Ligand Y-coated surface in an ELISA assay.
  • Ligand Y was coated onto 96-well plates at a concentration of 2 ⁇ g/mL diluted in PBS and incubated overnight followed by washing four times in PBS with 0.05% Tween-20. The plate was then blocked with PBS (Irvine Scientific, Santa Ana, CA) containing 0.5% (w/v) BSA (Sigma-Aldrich Corp., St. Louis, MO) for one hour at room temperature. In a separate plate, supernatants containing anti-Antigen E common light chain antibodies were diluted 1:10 in buffer. A mock supernatant with the same components of the antibodies was used as a negative control. Antigen E-mmH (described above) was added to a final concentration of 0.150 nM and incubated for one hour at room temperature.
  • PBS Irvine Scientific, Santa Ana, CA
  • BSA Sigma-Aldrich Corp., St. Louis, MO
  • the antibody/Antigen E-mmH mixture was then added to the plate containing Ligand Y and incubated for one hour at room temperature. Detection of Antigen E-mmH bound to Ligand Y was determined with Horse-Radish Peroxidase (HRP) conjugated to anti-Penta-His antibody (Qiagen, Valencia, CA) and developed by standard colorimetric response using tetramethylbenzidine (TMB) substrate (BD Biosciences, San Jose, CA) neutralized by sulfuric acid. Absorbance was read at OD450 for 0.1 sec. Background absorbance of a sample without Antigen E was subtracted from all samples. Percent blocking was calculated by division of the background-subtracted MFI of each sample by the adjusted negative control value, multiplying by 100 and subtracting the resulting value from 100.
  • HRP Horse-Radish Peroxidase
  • TMB tetramethylbenzidine
  • Tables 7 and 8 show the percent blocking for all 98 anti-Antigen E common light chain antibodies tested in the ELISA assay. ND: not determined under current experimental conditions.
  • K D Equilibrium dissociation constants for selected antibody supernatants were determined by SPR (Surface Plasmon Resonance) using a BIAcoreTM T100 instrument (GE Healthcare). All data was obtained using HBS-EP (10mM Hepes, 150mM NaCl, 0.3mM EDTA, 0.05% Surfactant P20, pH 7.4) as both the running and sample buffers, at 25°C. Antibodies were captured from crude supernatant samples on a CM5 sensor chip surface previously derivatized with a high density of anti-human Fc antibodies using standard amine coupling chemistry.
  • the binding affinities of common light chain antibodies comprising the rearrangements shown in Tables 3 and 4 vary, with nearly all exhibiting a K D in the nanomolar range.
  • the affinity data is consistent with the common light chain antibodies resulting from the combinatorial association of rearranged variable domains described in Tables 3 and 4 being high-affinity, clonally selected, and somatically mutated.
  • the common light chain antibodies described in Tables 3 and 4 comprise a collection of diverse, high-affinity antibodies that exhibit specificity for one or more epitopes on Antigen E.
  • Selected anti-Antigen E common light chain antibodies were tested for their ability to bind to the ECD of Antigen E and Antigen E ECD variants, including the cynomolgous monkey ortholog ( Mf Antigen E), which differs from the human protein in approximately 10% of its amino acid residues; a deletion mutant of Antigen E lacking the last 10 amino acids from the C-terminal end of the ECD (Antigen E- ⁇ CT); and two mutants containing an alanine substitution at suspected locations of interaction with Ligand Y (Antigen E-Ala1 and AntigenE-Ala2).
  • the Antigen E proteins were produced in CHO cells and each contained a myc-myc-His C-terminal tag.
  • Antigen E ECD protein or variant protein (described above) from 1 mL of culture medium was captured by incubation for 2 hr at room temperature with 1 x 10 6 microsphere (LuminexTM) beads covalently coated with an anti-myc monoclonal antibody (MAb 9E10, hybridoma cell line CRL-1729TM; ATCC, Manassas, VA). The beads were then washed with PBS before use. Supernatants containing anti-Antigen E common light chain antibodies were diluted 1:4 in buffer and added to 96-well filter plates. A mock supernatant with no antibody was used as negative control.
  • the beads containing the captured Antigen E proteins were then added to the antibody samples (3000 beads per well) and incubated overnight at 4°C. The following day, the sample beads were washed and the bound common light chain antibody was detected with a R-phycoerythrin-conjugated anti-human IgG antibody.
  • the fluorescence intensity of the beads (approximately 100 beads counted for each antibody sample binding to each Antigen E protein) was measured with a LuminexTM flow cytometry-based analyzer, and the median fluorescence intensity (MFI) for at least 100 counted beads per bead/antibody interaction was recorded. Results are shown in Tables 11 and 12.
  • the anti-Antigen E common light chain antibody supernatants exhibited high specific binding to the beads linked to Antigen E-ECD.
  • the negative control mock supernatant resulted in negligible signal ( ⁇ 10 MFI) when combined with the Antigen E-ECD bead sample, whereas the supernatants containing anti-Antigen E common light chain antibodies exhibited strong binding signal (average MFI of 2627 for 98 antibody supernatants; MFI > 500 for 91/98 antibody samples).
  • the relative binding of the antibodies to the variants were determined. All four Antigen E variants were captured to the anti-myc LuminexTM beads as described above for the native Antigen E-ECD binding studies, and the relative binding ratios (MFI variant /MFI Antigen E-ECD ) were determined.

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Claims (14)

  1. Maus, umfassend:
    (a) eine Ersetzung am endogenen Maus-K-Immunglobulin-leichte-Kette-variable-Region-Locus von endogenen Maus-κ-Immunglobulin-leichte-Kette-variable-Region-Gensegmenten mit einem einzelnen umgestellten humanen Vκ1-39/Jκ-Gensegment oder einem einzelnen umgestellten humanen Vκ3-20/Jκ-Gensegment, wobei das humane Gensegment mit einem endogenen Maus-κ-konstant-Gen operativ verknüpft ist, und wobei der Maus ein endogener Maus-κ-Immunglobulin-leichte-Kette-variable-Region-Locus fehlt, welcher in der Lage ist, sich umzustellen, und ein Gen zu bilden, welches eine Maus-κ-variable Region codiert; und
    (b) eine Ersetzung von 90-100% des endogenen Maus-schwere-Kette-variable-Region-Gen-Locus mit einer Vielzahl von humanen Schwere-Kette-variable-Region-Gensegmenten, wobei die humanen Schwere-Kette-variable-Region-Gensegmente mit einem endogenen Maus-schwere-Kette-konstant-Gen operativ verknüpft sind, und die humanen Schwere-Kette-variable-Region-Gensegmente in der Lage sind, sich umzustellen, und ein umgestelltes chimäres Mensch/Maus-schwere-Kette-Gen zu bilden.
  2. Maus gemäß Anspruch 1, wobei:
    (a) die Maus ferner einen Maus-κ-Intron-Enhancer 5' bezüglich der Maus-leichte-Kette-konstante-Region umfasst, oder
    (b) die Maus ferner einen Maus-κ-3'-Enhancer umfasst.
  3. Maus gemäß Anspruch 2, wobei:
    (a) die Vielzahl von humanen Schwere-Kette-variable-Region-Gensegmenten ein Segment, ausgewählt aus der Gruppe bestehend aus den humanen Immunglobulin-variable-Region-Gensegmenten 1-2, 1-8, 1-24, 2-5, 3-7, 3-9, 3-11, 3-13, 3-15, 3-20, 3-23, 3-30, 3-33, 3-48, 4-31, 4-39, 4-59, 5-51 und 6-1, und einer Kombination davon, umfasst,
    (b) die Vielzahl von humanen Schwere-Kette-variable-Region-Gensegmenten ein Segment, ausgewählt aus der Gruppe bestehend aus den Gensegmenten D1-7, D1-26, D3-3, D3-10, D3-16, D3-22, D5-5, D5-12, D6-6, D6-13 und D7-27, und einer Kombination davon, umfasst,
    (c) die Maus eine B-Zelle umfasst, welche eine umgestellte Immunglobulin-schwere-Kette-variable-Region-Gensequenz umfasst, umfassend ein humanes Schwere-Kette-variable-Region-Gen, abgeleitet aus einem VH-Gensegment, ausgewählt aus der Gruppe bestehend aus VH2-5, VH3-23, VH3-30, VH4-39, VH4-59, und VH5-51, und abgeleitet von einem D-Gensegment, ausgewählt aus der Gruppe bestehend aus D1-7, D1-26, D3-3, D3-16, D3-10, D3-22, D5-5, D5-12, D6-6, D6-13 und D7-27, oder
    (d) die Maus eine B-Zelle umfasst, welche eine umgestellte Immunglobulin-schwere-Kette-variable-Region-Gensequenz umfasst, welche ein VH-, JH- und DH-Gensegment umfasst, ausgewählt aus der Gruppe bestehend aus VH 2-5 + JH 1 + D6-6, VH3-23 + JH4 + D3, VH3-23 + JH4 + D3-10, VH3-30 + JH 1 + D6-6, VH3-30 +JH3 + D6-6, VH3-30 + JH4 + D1 -7, VH3-30 + JH4 + D5-12, VH3-30 + JH4 + D6-13, VH3-30 + JH4 + D6-6, VH3-30 + JH4 + D7-27, VH3-30 + JH5 + D3-22, VH3-30 + JH5 + D6-6, VH3-30 + JH5 + D7-27, VH4-39 + JH3 + D1 -26, VH4- 59 + JH3 + D3-16, VH4-59 + JH3 + D3-22, VH4-59 + JH4 + D3-16, VH5-51 + JH3 + D5-5, VH5-51 + JH5 + D6-13 und VH5-51 + JH6 + D3-16.
  4. Maus gemäß Anspruch 1, wobei:
    (a) das humane Vκ1-39-Gensegment in einer Umstellung mit einem humanen Jκ5-Gensegment vorliegt, oder
    (b) das humane Vκ3-20-Gensegment in einer Umstellung mit einem humanen Jκ1-Gensegment vorliegt.
  5. Zelle, isoliert aus einer genetisch veränderten Maus gemäß einem der vorangehenden Ansprüche, wobei die Zelle umfasst:
    (a) eine Ersetzung am endogenen Maus-K-Immunglobulin-leichte-Kette-variable-Region-Locus von endogenen Maus-κ-Immunglobulin-leichte-Kette-variable-Region-Gensegmenten mit einem einzelnen humanen Vκ1-39/Jκ-Gensegment oder einem einzelnen humanen Vκ3-20/Jκ-Gensegment, wobei das humane Gensegment mit einem endogenen Maus-κ-konstant-Gen operativ verknüpft ist, und wobei der Zelle ein endogener Maus-κ-Immunglobulin-leichte-Kette-variable-Region-Locus fehlt, welcher in der Lage ist, sich umzustellen, und ein Gen zu bilden, welches eine Maus-κ-variable Region codiert, und,
    (b) eine Ersetzung von 90 bis 100% des endogenen Maus-schwere-Kette-variable-Region-Gen-Locus mit einer Vielzahl von humanen Schwere-Kette-variable-Region-Gensegmenten, wobei die humanen Schwere-Kette-variable-Region-Gensegmente mit einem endogenen Maus-schwere-Kette-konstant-Gen operativ verknüpft sind, und die humanen Schwere-Kette-variable-Region-Gensegmente in der Lage sind, sich umzustellen, und ein umgestelltes chimäres Mensch/Maus-schwere-Kette-Gen zu bilden.
  6. Nichttherapeutisches Verfahren zur Herstellung eines Antikörpers, welcher ein Antigen von Interesse bindet, umfassend Immunisieren einer Maus gemäß einem der Ansprüche 1 bis 4 mit einem Antigen von Interesse, Erhalten einer Immunglobulin-variable-Region-Gensequenz aus der Maus, und Ausnutzung der Immunglobulin-variable-Region-Gensequenz, um einen Antikörper zu produzieren, welcher das Antigen bindet.
  7. Verfahren gemäß Anspruch 6, welches ferner umfasst:
    - Exprimieren in einer einzigen Zelle: (a) eine erste Schwere-Kette-variable-Region (VH)-Gensequenz der Maus gemäß einem der Ansprüche 1 bis 4, welche immunisiert worden ist, wobei die VH-Gensequenz mit einer humanen Schwere-Kette-konstante-Region (CH)-Gensequenz fusioniert ist, und (b) eine Leichte-Kette-variable-Region (VL)-Gensequenz der Maus gemäß einem der Ansprüche 1 bis 4, welche immunisiert worden ist, wobei die VL-Gensequenz mit einer humanen Leichte-Kette-konstante-Region (CL)-Gensequenz fusioniert ist,
    - Halten der Zelle unter Bedingungen, die ausreichend sind, einen vollständig humanen Antikörper zu exprimieren, und
    - Isolieren des Antikörpers.
  8. Verfahren gemäß Anspruch 7, wobei die Zelle eine zweite VH-Gensequenz einer Maus gemäß einem der Ansprüche 1 bis 4 umfasst, welche immunisiert worden ist, wobei die VH-Gensequenz mit einer humanen CH-Gensequenz fusioniert ist, wobei die erste VH-Gensequenz eine VH-Domäne codiert, welche ein erstes Epitop erkennt, und die zweite VH-Gensequenz eine VH-Domäne codiert, welche ein zweites Epitop erkennt, wobei das erste Epitop und das zweite Epitop nicht identisch sind.
  9. Nichttherapeutisches Verfahren zur Herstellung eines humanen bispezifischen Antikörpers, wobei das Verfahren Immunisieren einer Maus gemäß einem der Ansprüche 1 bis 4, und Herstellen des bispezifischen Antikörpers mit Hilfe von humanen Variable-Region-Gensequenzen aus B-Zellen aus der Maus umfasst.
  10. Verfahren gemäß Anspruch 9, wobei das Verfahren umfasst:
    (a) Identifizieren eines klonal selektierten Lymphozyten der Maus gemäß einem der Ansprüche 1 bis 4, wobei die Maus immunisiert worden ist, und ihr erlaubt wurde, eine Immunantwort gegen ein Antigen von Interesse zu entwickeln, wobei der Lymphozyt einen Antikörper exprimiert, welcher spezifisch an das Antigen von Interesse bindet,
    (b) Erhalten einer Nucleotidsequenz, welche die humane Schwere-Kette-variable-Region des Antikörpers codiert, die spezifisch an das Antigen von Interesse bindet, aus dem Lymphozyten oder dem Antikörper, und,
    (c) Einsetzen der Nucleotidsequenz gemäß (b) zur Herstellung eines bispezifischen Antikörpers.
  11. Verfahren gemäß Anspruch 10, wobei die Schritte (a) bis (c) ein erstes Mal für ein erstes Antigen von Interesse durchgeführt werden, um eine erste humane Schwere-Kette-variable-Region-Sequenz zu erzeugen, und die Schritte (a) bis (c) ein zweites Mal für ein zweites Antigen von Interesse durchgeführt werden, um eine zweite humane Schwere-Kette-variable-Region-Sequenz zu erzeugen, und wobei die erste humane Schwere-Kette-variable-Region-Sequenz mit einer ersten humanen Schwere-Kette-konstante-Region fusioniert exprimiert wird, um eine erste humane schwere Kette zu bilden, die zweite humane Schwere-Kette-variable-Region-Sequenz mit einer zweiten humanen Schwere-Kette-konstante-Region fusioniert exprimiert wird, um eine zweite humane schwere Kette zu bilden, wobei die erste und die zweite humane schwere Kette in der Gegenwart einer einzigen humanen leichten Kette exprimiert werden, welche aus einem Vκ1-39- oder einem Vκ3-20-Gensegment exprimiert wird.
  12. Verfahren gemäß Anspruch 10, wobei das Verfahren umfasst:
    (a) Klonieren humaner Schwere-Kette-variable-Region-Sequenzen aus B-Zellen aus:
    - der Maus gemäß einem der Ansprüche 1 bis 4, welche mit einem ersten Antigen von Interesse immunisiert worden ist, und
    - derselben Maus, oder einer anderen Maus, welche genetisch dieselbe ist, welche mit einem zweiten Antigen von Interesse immunisiert worden ist,
    (b) Exprimieren der humanen Schwere-Kette-variable-Region-Sequenzen gemäß (a) mit derselben Schwere-Kette-konstante-Region und derselben leichten Kette in einer Zelle, um einen bispezifischen Antikörper herzustellen.
  13. Verfahren gemäß Anspruch 11 oder Anspruch 12, wobei die erste humane schwere Kette eine Modifikation umfasst, welche die Affinität der ersten humanen schweren Kette für Protein A eliminiert oder wesentlich verringert, und die zweite humane schwere Kette die Fähigkeit beibehält, Protein A zu binden, wobei die Modifikation, welche die Affinität der ersten humanen schweren Kette für Protein A eliminiert, oder wesentlich verringert, aus einer 95R (EU 435R), einer 96F (EU 436F), und einer Kombination davon ausgewählt wird.
  14. Verwendung einer Maus gemäß einem der Ansprüche 1 bis 4, um ein(e) Immunglobulin-schwere-Kette-variable-Domäne oder -Gensegment auszuwählen.
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